In the United States, 31 million people have hearing loss, which is typically due to the loss of hair cells in the inner ear, which are needed for norma hearing. Hearing loss is permanent in humans, since hair cells cannot be replaced. However, regeneration of hair cells readily occurs in birds, restoring hearing function. To develop hair cel regeneration as a therapy for hearing loss, we must understand the factors controlling hair cell regeneration in non-mammals. Many prior studies have focused on the role of Atoh1, a basic helix-loop-helix transcription factor, in control of hair cell production because Atoh1 is necessar for hair cell differentiation during development, and because Atoh1 is sufficient to force differentiation of ectopic hair cells in the developing and mature inner ear. Therefore, Atoh1 is an excellent candidate for promoting hair cell regeneration. However, very little is known about (1) Atoh1's specific functions during regeneration in non- mammals, or (2) how Atoh1 activity is controlled during quiescence and regeneration. Therefore, additional studies are required before Atoh1 can be validated as a target for hair cell therapy at the human level. This proposal tests the following related hypotheses: (1) BMPs antagonize Atoh1 activity and keep supporting cells quiescent under normal conditions while decreasing hair cell differentiation after damage; (2) inhibitor of differentiation (ID) transcription factors oppose Atoh1 in their control of supporting cell behaviors in control tissue and after hair cell damage; and (3) BMPs act to antagonize hair cell differentiation through increased ATOH1 protein degradation. Post-hatch chickens will be used to test these hypotheses in addition to organotypic cultures, gene misexpression tools, and histological analyses. These experiments will move the field closer to understanding how hair cell regeneration is regulated in chickens, which will pave the way for more informed, hypothesis-based experiments in mammalian models. This approach will be instrumental in identifying strong candidates for gene therapy for eventual application in humans. PUBLIC HEALTH RELEVANCE: The proposed study will investigate the mechanisms that chickens use to regenerate hair cells in the inner ear, which are necessary for normal hearing function. Because humans cannot regenerate hair cells, it is necessary to determine how the chicken regulates this complex task in order to eventually develop strategies for promoting hair cell regeneration in humans.